Reticulons are ubiquitous, highly conserved integral membrane proteins present in all eukaryotes. Reticulon proteins generate the structure of the peripheral endoplasmic reticulum (ER) by shaping the membrane bilayer into a network of tubules in eukaryotic cells. Despite the continuity of the ER membrane, which is composed of the nuclear envelope as well as peripheral ER sheets and tubules, these proteins localize exclusively to ER tubules. The overall goal of the proposed program is to develop our understanding of how the reticulon proteins partition into and shape the ER membrane bilayer into tubules. The specific aims of the program include structure/function studies of vertebrate and yeast reticulon proteins to determine how they localize to tubular ER and which of their structural features are required for their membrane shaping activities. We will mutate various regions of the reticulon protein to determine the effect on protein localization, oligomerization, diffusional mobility, and membrane curvature. For these studies, we will be using fluorescence microscopy, immunoprecipitations, fluorescence recovery after photobleaching, and electron microscopy with immunogold labeling. These experiments will reveal how these integral membrane proteins localize to and shape the membrane bilayer of an organelle, the ER. We propose to use high-resolution EM techniques to analyze the 3-D architecture and surface of ER tubules formed in vitro from Xenopus egg extract. The impetus for this work is to look for a reticulon scaffold around ER tubules that could explain how these proteins directly shape ER tubules of defined dimensions. However, even in the absence of discovering a reticulon scaffold on ER tubules, this work would be the first high-resolution structural analysis of ER tubules and could reveal a great deal about the morphological properties of this organelle. Finally, we will be addressing whether reticulon-generated tubular ER morphology is important for cellular differentiation. We are studying the differentiation of neurons, which causes the morphology of neuronal cells to change quite dramatically and become highly polarized. Neuronal differentiation results in the extension of axons and dendrites, long processes that we show here are filled with tubular ER stained with the reticulon protein. We will address whether increasing and decreasing reticulon protein levels affects neuronal differentiation and what functions of the tubular ER are necessary for axon/neurite growth. We will look for reticulon interacting factors that contribute to the growth, packaging, and organization of the tubular ER into the axon/neurite and analyze the structure of the ER within neurite/axon by electron microscopy. This work will address the functional relevance of organelle morphology to cellular differentiation.